Planar Cell Polarity (PCP) signaling polarizes cells in epithelial sheets along an axis orthogonal to their apical- basal axis. Most of our mechanistic understanding of PCP signaling derives from work in Drosophila. However, a range of medically important developmental defects and physiological processes in vertebrates are under its control. PCP in vertebrates appears to conserve much or all of control of PCP signaling, including neural tube closure defects, polycystic kidneys, conotruncal heart defects, deafness and situs inversus. PCP polarizes skin and hair, and underlies directed migration during wound healing and invasion and metastasis of malignant cells the mechanism uncovered in flies, motivating considerable interest in studying PCP both in Drosophila. Studies in Drosophila reveal a modular system controlling PCP. A 'global' module comprising Fat(Ft), Dachsous(Ds) and Four-jointed(Fj) converts opposing tissue-level expression gradients of Fj and Ds into subcellular asymmetry of intercellular Ft-Ds heterodimers, and could therefore align PCP with the tissue axes. A 'core' module amplifies molecular asymmetry, localizing proximal (Van Gogh and Prickle) and distal (Frizzled, Dishevelled[Dsh]) proteins to opposite sides of the cell and coordinating polarity between neighboring cells. Morphological responses to the resulting molecular asymmetry are tissue specific. We have identified a mechanism linking these modules in series, and in so doing, revealed a fascinating cell biological mechanism. The global components Ft and Ds, via novel junctional ultrastructures, organize and tether arrays of polarized apical microtubules (MTs) that serve as substrates for directional trafficking of Dsh, thereby introducing a directional bias to the core module. Directional trafficking of Dsh breaks symmetry to bias core protein polarization. A paradox was the observation that the relationship between the direction of the global gradients and the direction of core module polarization is not conserved between tissues. We found that the predominance of the Prickle(Pk) or Spiny-legs(Sple) isoform of the core PCP component Pk controls gradient interpretation in these tissues by determining the direction of gradient dependent MTs, and thus the direction of MT dependent Dsh trafficking. These results raise a number of fascinating questions that we address in this proposal. How do the global components organize apical MTs? We hypothesize that Ft and Ds recruit proteins that capture and organize MTs. We will investigate how Pk-Sple controls Ft-Ds dependent MT orientation. Our data indicate that the Ft- Ds mechanism functions partially redundantly with a second signal originating at the wing margin. Preliminary data indicate that Wnt4 serves as this other signal, also by organizing polarized MTs. How does this other signal function? Once MTs are established, we wish to understand whether Dsh vesicle production is directly linked to these structures, and to study the signals for internalization and the control of molecular motors. Finally, we will study the E3 Ubiquitin ligase Cul1, which we have identified as a regulator of Pk function.